The aim of the present study was to evaluate, in vivo, the potential of o,p′-DDT to disrupt the endocrine system of mature male tilapia. In particular, the possibility that o,p′-DDT effects were mediated directly via the estrogen receptor (ER). Compounds with known ability to bind to the ER were employed: estradiol to induce and tamoxifen to inhibit the estrogenic effects result of the activation of the ER. In addition, an aromatase inhibitor, 4-hydrxyandrostenedione (4-OHA), was used to assess the ability of o,p′-DDT to induce estrogenic effects in a surrounding of low estradiol concentration. The effects of estradiol and o,p′-DDT were studied alone or in the presence of tamoxifen or 4-OHA at the end of a 12-day period of exposure. The main endpoints measured were plasma alkaline-labile phosphorous (ALP; an indirect indicator of vitellogenin), estradiol, testosterone and o,p′-DDT. It was found that o,p′-DDT was able to induce the vitellogenesis (measured as plasma ALP increase) and decrease the circulating levels of estradiol and testosterone. Interestingly, o,p′-DDT kept this ability in whole fish with low concentrations of estradiol which would exclude endogenous estradiol as indirect mediator of the estrogenic effects induced by o,p′-DDT. In addition, the plasma concentration of o,p′-DDT, instead of that of estradiol, was closely related to the plasma ALP increase induced by o,p′-DDT. This indicates that o,p′-DDT could have directly activated the vitellogenesis. The antiestrogenic action of tamoxifen to inhibit the vitellogenesis and the decrease on plasma estradiol induced by o,p′-DDT indicates that o,p′-DDT can bind directly to the ER. In conclusion, this in vivo study shows that o,p′-DDT has the potential to disrupt the endocrine system and strongly supports that the estrogenic actions of o,p′-DDT involve binding to the ER.